Recent epidemiological studies strongly suggest that flavonoids, a large class of phytochemicals present in fruits, vegetables and beverages, have protective roles in human diseases, most prominently in coronary heart disease and cancer. A rapidly growing number of biochemical studies show plausible mechanisms for these effects. However, it is not clear if the dietary flavonoids, including their glycosides, can gain access to proposed cellular sites of action; their biological fate in humans remains essentially unknown. The studies in this project propose to help fill this essential information gap. Our general working hypothesis is that the bioavailability of flavonoids is a function of cell membrane penetrability, protein binding and presystemic metabolism, and varies markedly with the chemical structure of the flavonoid. In Aim 1 we will focus on flavonoid uptake by the human hepatoma cell line Hep G2 and fresh hepatocytes and the mechanism(s) involved. This will include studies of specific inhibitors of carrier-mediated transporters. It will also include determination of plasma protein binding of the flavonoids and its effect on membrane penetration. In Aim 2 we will determine the metabolism of the flavonoids, using Hep G2 cells as well as human liver homogenates. This will include HPLC isolation of metabolites and structure identification using atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) and high field nuclear magnetic resonance (NMR) spectrometry. The enzymes involved will be identified by use of selective chemical and antibody inhibitors as well as isoform-specific expression systems. In Aim 3 we will determine the ability of flavonoids to traverse the intestinal epithelial cell border, using the human Caco-2 cell line, grown as monolayers on permeable filters, and excised rat intestine, as models. As with the hepatocytes in Aim 1, we will determine the effect of chemical structure on membrane permeability and mechanism(s) of transport. These studies will also include P-glycoprotein-mediated efflux as well as metabolism as factors limiting net transport. In aim 4 we will test our in vitro observations directly in vivo, using pharmacokinetic approaches. In these studies we will in first hand seek to determine the bioavailability of flavonoid glycosides and their subsequent hydrolysis to aglycones in normal volunteers through plasma and urine determinations.